Genetic structure of Plasmodiophora brassicae populations virulent on clubroot resistant canola (Brassica napus)

Clubroot, caused by Plasmodiophora brassicae Woronin, is a significant threat to the canola (Brassica napus L.) industry in Canada. Clubroot resistance has been overcome in more than 200 fields since 2013, representing one of the biggest challenges to sustainable canola production. The genetic structure of 36 single-spore isolates derived from 12 field isolates of P. brassicae collected before and after the introduction of clubroot resistant (CR) canola cultivars (2005-2014) was evaluated by simple sequence repeat (SSR) marker analysis. Polymorphisms were detected in 32 loci with the identification of 93 distinct alleles. A low level of genetic diversity was found among the single-spore isolates. Haploid linkage disequilibrium and number of migrants suggested that recombination and migration were rare or almost absent in the tested P. brassicae population. A relatively clear relationship was found between the genetic structure and virulence phenotypes of the pathogen as defined on the differential hosts of Somé et al., Williams and the Canadian Clubroot Differential (CCD) set. Although genetic variability within each pathotype group, as classified on each differential system, was low, significant genetic differentiation was observed among the pathotypes. The highest correlation between genetic structure and virulence was found among matrices produced with genetic data and the hosts of the CCD set, with a threshold index of disease of 50% to distinguish susceptible from resistant reactions. Genetically homogeneous single-spore isolates provided a more complete and clearer picture of the population genetic structure of P. brassicae, and the results suggest some promise for the development of pathotype-specific primers.

Virulence and Molecular Diversity in the Kabatiella zeae Population Causing Maize Eyespot in China

Maize eyespot, caused by Kabatiella zeae, has become a major yield-limiting factor in maize planting areas in northeast China. Limited information is available on pathotypes, virulence, and the genetic diversity of the K. zeae population. We analyzed virulence and genetic diversity of 103 K. zeae isolates collected from six provinces in China with differential hosts and the amplified fragment length polymorphism (AFLP) technique, respectively. To evaluate the virulence, 103 isolates were inoculated on nine differential hosts (maize inbred lines)—E28, Shen137, Qi319, B73, Danhuang34, Zi330, Mo17, Huangzaosi, and CN165—and grouped into 23 pathotypes and three virulence groups according to the coded triplet nomenclature system on differential hosts. AFLP analysis resolved the set of isolates into four genetic diversity clusters (DICE similarity values of 76%). Genetic variation of K. zeae among and between pathotypes revealed that the pathogen population had a high genotypic diversity. The correlation between pathotypes, virulence, and genetic diversity grouping was low. A correlation between AFLP groups and geographic locations was detected.

Most Plasmodiophora brassicae Populations in Single Canola Root Galls from Alberta Fields are Mixtures of Multiple Strains

Clubroot, caused by Plasmodiophora brassicae, is an important disease on canola in Alberta, Canada. The pathogen is grouped into pathotypes according to their virulence reaction on differential hosts. Multiple pathotypes or strains are known exist in one field, one plant, or even one gall. This study was conducted with the objective of testing the prevalence of the coexistence of multiple strains in a single gall. In all, 79 canola clubroot galls were collected from 22 fields across Northern Alberta in 2018. Genomic DNA extracted from these single galls was analyzed using RNase H-dependent PCR (rhPCR). The rhPCR primers were designed to amplify a partial sequence of a dimorphic gene, with one primer pair specific to one sequence and the other primer pair specific to the alternative sequence. The amplification of both sequences from DNA obtained from a single gall would indicate that it contains two different P. brassicae strains. The rhPCR analyses indicated that the P. brassicae populations in 50 of the 79 galls consisted of more than one strain. This result emphasizes the need for cautious interpretation of results when a single-gall population is subject to pathotyping or being used as inoculum in plant pathology research. It also confirms that the maintenance of pathotype diversity within single root galls is a common occurrence which has implications for the durability, and stewardship, of single-gene host resistance.

Pathotype diversity of Fusarium oxysporum f. sp. mungcola causing wilt on mungbean (Vigna radiata)

Fusarium wilt, caused by Fusarium oxysporum f. sp. mungcola (Fom), is an increasingly serious disease of mungbean (Vigna radiata (L.) R.Wilczek) in China. Pathogenic variability has been observed among Fom isolates; however, there are no reports describing Fom races or pathotypes. Thus, this study was conducted with the aim of developing a set of pathotype differentials to reveal Fom pathotype diversity by assessing virulence variability of Fom isolates. First, 105 mungbean cultivars were screened against a standard virulent Fom isolate (F08). Eleven of the 105 cultivars were selected as candidate differentials of Fom according to resistance phenotype and genetic background. Second, the resistance of the 11 candidate differential cultivars was tested against 30 Fom isolates from different geographical origins in China. Highly significant differences were observed among isolate × cultivar interaction patterns, indicating that pathotype differentiation exists in Fom isolates. Based on the different reaction patterns combining with genetic background, seven of the 11 cultivars were selected to constitute a set of differential hosts of Fom pathotype, used to distinguish pathotypes of 84 Fom isolates from different geographical regions by evaluating the virulence reaction pattern. The results showed that the 84 Fom isolates were defined as 12 pathotypes. Finally, we tried to confirm whether the 12 Fom pathotypes could be distinguished by a PCR-based diagnostic method based on the two genes (SIX6 and SIX11) reported to be associated with Fom pathogenicity. However, the Fom pathotype could not be distinguished by variation of the PCR products or their resulting sequences of the two genes. This is the first study to develop a set of Fom pathotype differential hosts and identify 12 Fom pathotypes, which provides important information for resistance breeding and disease control.

Identification of temperature sensitive resistance to Puccinia striiformis f. sp. tritici in wheat differential hosts and its potential applications in resistance breeding

Temperature affects wheat resistance responses infected by Puccinia striiformis f. sp. tritici (Pst). In order to identify if thirty-one entries of Chinese, international and other tester wheat cultivars possess temperature sensitive resistance, the entries were studied in seedling stage at two different day/night temperature regimes (24/18°C and 14/10°C). Four entries, Lutescens 128, Funo, Lee and Carstens V, were confirmed no temperature sensitive resistance genes. Six wheat cultivars, Early Piemium, Fengchan 3, Fulhard, Heines VII, Mentana and Virgilio, have shown temperature sensitive resistance. Comparison with four standard lines (S110, S111, S112, S113) with 0-3 temperature sensitive genes, derived from crosses of Itana/PI 178383, the resistance to Pst race 10E162 in Virgilio controlled by two temperature sensitive genes, and Mentana and Fulhard each possessed one temperature sensitive gene. Virgilio, Fulhard and Mentana as the temperature sensitive gene resources are useful in breeding for resistance to stripe rust. As the differential hosts of wheat stripe rust, it is necessary to strictly control the temperature without exceeding 18°C, since infection type may differ due to the different temperature. Keywords: Differential hosts; Puccinia striiformis f. sp. tritici; Temperature sensitive resistance; Wheat

Virulence, Molecular Diversity, and Mating Type of Curvularia lunata in China

Curvularia leaf spot (CuLS), caused by Curvularia lunata, is a devasting foliar disease in the maize-growing regions of China. Resistant varieties were widely planted in these regions in response to CuLS. However, over time, C. lunata has gradually adapted to the selective pressure and, in recent years, the incidence of CuLS has increased. To assess the correlation between virulence and genetic diversity, a total of 111 isolates was collected from 15 maize-growing regions located in nine provinces in China. These isolates were evaluated for virulence on maize using nine differential hosts: Shen135, CN165, Mo17, Luyuan92, 78599, Ye478, B73, E28, and Huangzaosi. To evaluate the genetic diversity, 657 polymorphic amplified fragment length polymorphism markers were generated. Results showed that the isolates could be grouped into three pathotypes according to the phenotypic expression of the differential inbred lines. Isolates were clustered into two genetic diversity groups and further divided into subgroups. However, the correlation between virulence and genetic diversity grouping was low. Also, there was a low correlation observed between pathotype and geographic distribution. The ratio of mating type I to mating type II for all isolates was close to 3:4.

Specific Genes Identified in Pathotype 4 of the Clubroot Pathogen Plasmodiophora brassicae

Clubroot is an important disease of cruciferous crops caused by Plasmodiophora brassicae, and pathotypes are classified based on the response of differential hosts. This study was conducted to identify genetic markers able to differentiate pathotypes. Differential expression of genes between pathotype 4 (P4) and pathotype 7 (P7) was assessed according to transcriptome data of molecular marker screening. Among the pathotypes (P2, P4, P5, P7, P9, P10, and P11) tested, six genes were exclusive to P4, dividing the isolates into three types: PBRA_003263 and PBRA_003268 were present in all P4 isolates, PBRA_000003/Novel512 were found in a type of P4 (P4-1), and Novel137/PBRA_005772 were found in another P4 type, P4-2. Amplicons for all six genes were produced for only one isolate, which we named P4-3. This study is the first to establish a molecular identification system for P4 the, predominant pathotype in China. The genes identified might serve as molecular markers for differentiation of P4 from other pathotypes and may also distinguish different types of P4.

Characterization of Five Molecular Markers for Pathotype Identification of the Clubroot Pathogen Plasmodiophora brassicae

Clubroot disease is an important disease on cruciferous crops caused by Plasmodiophora brassicae infections. The pathotypes have been classified based on the reactions of differential hosts. However, molecular markers of particular pathotypes for P. brassicae are limited. In this study, we found five genetic markers in association with different pathotypes. Different gene expression patterns among different pathotypes (P4, P7, P9, and P11) were assayed according to the transcriptome data. The assay indicated that molecular markers PBRA_007750 and PBRA_009348 could be used to distinguish P11 from P4, P7, and P9; PBRA_009348 and Novel342 could distinguish P9 from P4, P7, and P11; and PBRA_008439 and Novel342 could represent a kind of P4. Polymerase chain reaction cycles ranging from 25 to 30 were able to identify the predominant pathotype in general. Therefore, these molecular markers would be a valuable tool to identify and discriminate pathotypes in P. brassicae population.

Pathogenic variability in Exserohilum turcicum and identification of resistant sources to turcicum leaf blight of maize (Zea mays L.)

Turcicum leaf blight of maize incited by Exserohilum turcicum (Pass.) Leonard and Suggs is the major limiting factor of maize production in temperate agro-ecologies. Disease management through host plant resistance is the most effective strategy. In the present study among 26 maize genotypes which were initially screened for resistance against E. turcicum under field conditions, 8 genotypes viz., PS 39, CML 451, CML 470, CML 472, VL 1030, VL 1018140, VL1018527 and SMI178-1 were found resistant when screened against twelve isolates of E. turcicum under artificial epiphytotic conditions. Eight genotypes viz., PS45, CML165, CML459, VL1249, VL0536, SMC-5, SMC-3 and KDL 211 were found moderately resistant with disease grade ranged from 2.1-2.5. These maize genotypes possess resistance to turcicum leaf blight can be used successfully in developing high yielding early maturing varieties for high altitude temperate agro-ecologies. The fungus E. turcicum is highly variable in nature. Variability studies on pathogenicity were conducted on twelve isolates of E. turcicum on eleven putative differential maize lines. During the present study a wide pathogenic variation was observed among the twelve isolates of E. turcicum. Cluster analysis on the basis of similarity or dissimilarity in reaction types exhibited by the differential hosts, clustered the isolates into 6 pathogenic groups. The isolates belonged to higher altitudes (Kti 10, Kti11, Kti5) were found to be more aggressive as compared to the isolates of low altitude areas.

Characterization of a Gene Identified in Pathotype 5 of the Clubroot Pathogen Plasmodiophora brassicae

Clubroot caused by Plasmodiophora brassicae is an important disease of crucifers worldwide. Isolates of the pathogen can be classified into pathotypes according to their pathogenicity on differential hosts. In this study, the presence or absence of all database-available nonhousekeeping P. brassicae genes (118 in total) were assessed by polymerase chain reaction (PCR) analysis in isolates belonging to five P. brassicae pathotypes (2, 3, 5, 6, and 8 according to Williams’ differential set). One gene, designated Cr811, was present exclusively in the isolate of pathotype 5. This was further confirmed by dot blot hybridization and by PCR using alternative DNA preparations and primers. Reverse transcription quantitative PCR analysis indicated that in planta expression of Cr811 was up-regulated during canola infection, especially in the stage of secondary plasmodia. Primers specific to Cr811 could distinguish a field isolate of P. brassicae belonging to pathotype 5 from two other field isolates representing pathotypes 3 and 8. These findings suggest that Cr811 is a gene that is potentially involved in clubroot pathogenesis and that it also might serve as a molecular marker for differentiation of pathotype 5 from other pathotypes.